MiRNAs Mediate The Cross-talk Of RNA Interference And RNA Splicing By Facilitating The Formation Of AGO2-hnRNP U Complex In The Nuclei

Gene silencing,also known as RNA interference,is a significant molecular mechanism,which functions in controlling gene expression in eukaryotic cells.In eukaryotic cells,small RNAs guide Argonaute(Ago)proteins to m RNAs establishing the RNA-induced silencing complex(RISC)and bind to target RNAs according to the base complementary matching principle.Ago proteins take advantage of the hydrolase activity to degrade or translation inhibit of their target m RNAs.AGO2 is the only member of mammalian Ago protein family which possesses the catalytic activity and plays a key role in regulating gene expression.In recent years,researchers reported that AGO2 also functions in the nuclei as well as cytoplasm,which is the classical location of gene silencing.It is known that the nuclear AGO2 are involved in DNA double-strand break repair,DNA methylation and RNA alternative splicing.However,the concrete mechanisms in which nuclear AGO2 participate have not been fully elucidated.We found out AGO2-interacted proteins with nuclear extracts by co-immun oprecipitation(Co-IP)assays and identified a cluster of proteins involved in alternative splicing by Mass Spectrometry(MS)and GO analyses.hnRNP U is a member of this classification.RNA alternative splicing is a specific and general gene regulation mechanism of eukaryote.Spliceosome produces different transcripts with a same pre-m RNA by different processing ways,which is a central reason of the protein diversity of metazoan.hnRNP protein family,which participate in splicing in complicated and flexible ways,is a significant splicing regulated protein family.hnRNP U is the maximal molecular weight member of the hnRNP protein family and plays an important role in regulating splicing by affecting the maturation of U2 sn RNP,which is a member of spliceosome.We demonstrated that AGO2 interacted with hnRNP U by Co-IP assays with nuclear extracts and the two proteins presented nuclei foci in the immunofluorescence experiments.We constructed prokaryotic expression vector,GST-AGO2 and His-hnRNP U.However,recombinant protein His-hnRNP U could not be pulled-down by GST-AGO2,indicating that they might interact indirectly by certain bridge factors.We further performed Co-IP assays using RNase A-treated nuclear extracts and the results showed that AGO2 interacted with hnRNP U in an RNA-dependent manner.We constructed the mutant AGO2-Y529 E,which could not bind to small RNAs.The mutant had no interaction with hnRNP U,demonstrating that small RNAs promoted the formation of functional AGO2-hnRNP U complex.The overexpressing or knocking down assays showed that hnRNP U or AGO2 reciprocally reduced protein level.Treating with cycloheximide(CHX)and proteasomal inhibitor MG132 implied that the degradation resulted from the proteasome pathway.hnRNP U induced degradation of AGO2 both in the nuclei and cytoplasm that was improved by detecting AGO2 protein level with nuclear or cytoplasmic extracts.In consideration of the nuclear location of hnRNP U,we found out four nuclear target RNAs by consulting literature and web forecast.The RNA levels of nuclear targets were affected by overexpressing hnRNP U and supplying AGO2 verified that hnRNP U regulated nuclear RNAi through reducing protein level of AGO2.We found out two novel splicing targets(ZFY and GADD45A)of hnRNP U.The effect of the target splicing demonstrated that AGO2 regulated alternative splicing by affecting the protein level of hnRNP U.The further assays showed that the capacity of regulating splicing of AGO2 was related to the binding capacity of small RNA.Small RNAs participated in splicing by not only mediating the interaction between AGO2 and hnRNP U in hnRNP U-dependent manner but also promoting the binding of AGO2 and target RNAs in hnRNP U-independent manner.Furthermore,the results showed that the nuclei-cytoplasm distribution of AGO2 affected splicing.The high-throughput sequencing result suggested that AGO2 affected the splicing events of 83.4% genes and 76.5% of them were associated with small RNAs.We sought out the co-interactive RNAs between AGO2 and hnRNP U utilizing RIP-seq assay and the bioinformatics analysis results determined the corresponding mi RNA to the co-interactive RNAs.To our excitement,the portion of mi RNAs was highly consistent with the mi RNAs corresponding to the splicing targets,which were associated with small RNAs.We demonstrated that the highly consistent 9 mi RNAs affected alternative splicing and some of them regulated splicing in hnRNP U-dependent manner.The common 9mi RNAs showed the highly consensus sequence at 3' and 5' end and exhibited common characteristics in regulating splicing.We next proved that DOX affected the splicing of target genes by inhibiting AGO2 exporting from nuclei.The influenced splicing by DOX,which was resulted in functional transcripts increase or decrease,provided evidence to the inhibitory effect of tumor of DOX.The data bank showed that the protein expression level of AGO2 and hnRNP U exhibited negative correlation in many cell lines.We detected the protein level with four cell lines and the tumor cell lines showed high AGO2 protein level and low hnRNP U protein level.The normal cell lines showed high hnRNP U protein level and low AGO2 protein level.This phenomenon produced an effect on different transcripts leading to an induced or reduced RNA level of neoplastic transcripts,which affected tumorigenesis.In summary,this article proceeded from the nuclear function of AGO2 and focused on the relationship between nuclear RNAi and alternative splicing.The bioinformatics analysis combining with molecular biology experiments proved that small RNAs participated in splicing and found out concrete splicing target genes and specific mi RNAs,which regulated the splicing of targets.Our full text explained the mechanism of DOX inhibitory effect of tumor in a point of view of regulating splicing.